Field of the Invention
The present invention relates generally to skin regeneration and wound healing, and more specifically, to small molecule-mediated induction of dermal human fibroblast proliferation and increased protein production.
Background Information
The skin, the largest organ of the body in vertebrates, is composed of the epidermis and dermis with a complex nerve and blood supply. A third layer, the hypodermis, is composed mainly of fat and a layer of loose connective tissue. These three layers play an important role in protecting the body from any mechanical damage such as wounding.
The dermis, situated directly below the epidermis, constitutes the bulk of the skin and is composed of collagen with some elastin and glycosaminoglycans (GAGs). The major cell type present in the dermis is fibroblast, which is capable of producing remodeling enzymes such as proteases and collagenases that play an important role in the wound healing process.
Wound healing is a sequential mechanism and it is more specifically an event-driven process, whereby signals from one cell type set off cascades in other cell types, which propel the wound through the phases of healing. Adult wound healing is essentially a repair process, which normally exhibits scarring. Tissue repair begins immediately with fibrin clot deposition at the site of injury, preventing hemorrhage from damaged blood vessels. Circulating platelets then aggregate at the site of injury and various inflammatory mediators, such as PDGF, TGF-α and TGF-β, epidermal growth factor (EGF) and FGFs, are released. These molecules are also believed to play major roles downstream in the wound repair process.
The inflammatory response of adult tissues to wounding is characterized by an early influx of neutrophils whose numbers steadily increase and reach a maximum 24-48 hours post-wounding. As the neutrophil numbers begin to decline, macrophages take over and repopulate the wound site. Re-epithelialization also occurs at the same time, with keratinocytes migrating across the granulation tissue from deep within the dermis and the basal cells of the wound edge. As soon as the keratinocytes have re-established the barrier property of the skin, they resume a basal cell phenotype upon contact inhibition and differentiate into a stratified squamous keratinizing epidermis.
The final phases of the inflammatory response and epithelialization coincide with the migration of fibroblasts and endothelial cells and the formation of granulation tissue. Angiogenesis and fibroplasia then take place, with fibroblasts becoming the dominant cell type, laying down collagen and ECM. Remodeling of collagen occurs using matrix metalloproteinases produced by the fibroblasts and macrophages.
Dermal fibroblasts require far higher concentrations of fibroblast growth factor (FGF) in order to undergo cell replication and are responsible for creating the ECM, which organizes the stratified squamous epithelial cells of the epidermis into a unified tissue. Furthermore, dermal fibroblasts create long fibrous bands of connective tissue which anchor the skin to the fascia of the body. Without dermal fibroblasts, a wound site cannot regenerate extracellular matrix and epidermis skin cells cannot proliferate over the wound site. Therefore, without dermal fibroblasts the skin cannot properly recover from injury.
In humans, skin represents approximately one-tenth of the body mass, and damage such as trauma, disease, burn or surgery to a part of this major organ has dramatic consequences. Tissue-engineered skin substitutes are used in combating acute and chronic skin wounds. Tissue engineering aims to regenerate new biological material for replacing diseased or damaged tissues or organs. To achieve this, not only is a source of cells required, but also an artificial extracellular matrix (ECM) upon which the cells can be supported.
Natural biopolymers such as collagen and fibronectin have been investigated as potential sources of biomaterial to which cells can attach. The first generation of degradable polymers used in tissue engineering were adapted from other surgical uses and have drawbacks in terms of mechanical and degradation properties. This has led to the development of synthetic degradable gels primarily as a way to deliver cells and/or molecules in situ, the so-called smart matrix technology. Tissue or organ repair is usually accompanied by fibrotic reactions that result in the production of a scar. Certain mammalian tissues, however, have a capacity for complete regeneration without scarring; good examples include embryonic or fetal skin and the ear of the MRL/MpJ mouse. Investigations of these model systems revealed that in order to achieve such complete regeneration, the inflammatory response is altered such that the extent of fibrosis and scarring is diminished.
Dermal fibroblasts are a dynamic population of cells that have a key role in ECM deposition, epithelial-mesenchymal interactions and wound healing. Fibroblasts are readily cultured in the laboratory and incorporation of fibroblasts into tissue-engineered skin substitutes has produced encouraging results including symptomatic pain relief, more rapid healing of acute and chronic wounds, less scarring and better cosmetic results. However, at the present time, there are no models of bioengineered skin that completely replicate the anatomy, physiology, biological stability or aesthetic nature of uninjured skin.
Various tissue-engineered skin products need to be compared in multicenter clinical trials as this would enable the identification of specific products for particular clinical applications. This could potentially lead to a reduction in their costs following increased use of the specified product. In addition, a combination of tissue-engineered skin substitutes with cytokines and growth factors may in the future be used to enhance wound healing as well as afford the possibility of incorporating defensins for antimicrobial benefit. A need therefore exists for novel approaches to generate a skin replacement whose materials technology is based not only upon intelligent design, but also upon the molecules involved in the process of regeneration.